This grant is in response to PAR-12-058, Solicitation of Assays for High Throughput Screening to Discover Chemical Probes. Platelets play a critical role in thrombosis, the major cause of mortality and morbidity. Thrombin is present during neurotrauma and activates protease activated receptors, which causes oxidative stress and inflammation locally. PAR4 expression is increased under these conditions, which could lead to serious neurodegenerative sequelae. Medical management of acute coronary syndrome and cerebrovascular injury is centered on anti-platelet therapies. Currently available therapies, aspirin, clopidogrel, and ?IIb?3 inhibitors, do not fully attenuate platelet activation, can have delayed onset and long lived effects, and are linked to adverse bleeding events. Thrombin receptor antagonists (TRAs) are highly awaited in clinical medicine but Vorapaxar, a TRA that targets PAR1, was recently shown in Phase III clinical trials to increase clinically significant bleeding including intracranial hemorrhage. Because of PAR4's low affinity for thrombin, it is activated locally at the site of the 3-dimensional clot as more thrombin builds up. Because of the delay in activation we hypothesize that PAR4 antagonism might not affect hemostasis as potently and thus may be a better therapeutic target than PAR1 in thrombosis and cerebrovascular injury. Inhibition of PAR4 would not perturb signaling through the PAR1 receptor, which is essential for basic hemostasis during injury. Our major hypothesis is that inhibition of PAR4 is a potential target for platelet inhibition in thrombosis and cerebrovascular injury. Due to the lack of tool compounds, the field's understanding of the role of PAR4 in physiological environments is limited. To address these questions, we will 1) screen for negative allosteric modulators/competitive antagonists of PAR4. These compounds will avoid the difficulty that comes from trying to displace the tethered ligand which is present at molar concentrations. We will build on Vanderbilt's many successes in generating novel ligands that target the allosteric sites of GPCRs by running a triple add HTS screen, 2) we will identify compounds which inhibit thrombin stimulation of PAR4 and are selective for PAR4 in CHO cells and human platelets which express PAR4 endogenously, 3) we will determine whether the mechanism of inhibition is competitive or non-competitive using Schild analysis. These studies should provide valuable tool compounds which would have advantages of temporal and spatial specificity, becoming efficacious only when thrombin is present.